Structural alterations and inflammation in the heart after multiple trauma followed by reamed versus non-reamed femoral nailing

Abstract

Background: Approximately 30,000 patients with blunt cardiac trauma are recorded each year in the United States. Blunt cardiac injuries after trauma are associated with a longer hospital stay and a poor overall outcome. Organ damage after trauma is linked to increased systemic release of pro-inflammatory cytokines and damage-associated molecular patterns. However, the interplay between polytrauma and local cardiac injury is unclear. Additionally, the impact of surgical intervention on this process is currently unknown. This study aimed to determine local cardiac immunological and structural alterations after multiple trauma. Furthermore, the impact of the chosen fracture stabilization strategy (reamed versus non-reamed femoral nailing) on cardiac alterations was studied.

Experimental approach: 15 male pigs were either exposed to multiple trauma (blunt chest trauma, laparotomy, liver laceration, femur fracture and haemorrhagic shock) or sham conditions. Blood samples as well as cardiac tissue were analysed 4 h and 6 h after trauma. Additionally, murine HL-1 cells were exposed to a defined polytrauma-cocktail, mimicking the pro-inflammatory conditions after multiple trauma in vitro.

Results: After multiple trauma, cardiac structural changes were observed in the left ventricle. More specifically, alterations in the alpha-actinin and desmin protein expression were found. Cardiac structural alterations were accompanied by enhanced local nitrosative stress, increased local inflammation and elevated systemic levels of the high-mobility group box 1 protein. Furthermore, cardiac alterations were observed predominantly in pigs that were treated by non-reamed intramedullary reaming. The polytrauma-cocktail impaired the viability of HL-1 cells in vitro, which was accompanied by a release of troponin I and HFABP.

Discussion: Multiple trauma induced cardiac structural alterations in vivo, which might contribute to the development of early myocardial damage (EMD). This study also revealed that reamed femoral nailing (reamed) is associated with more prominent immunological cardiac alterations compared to nailing without reaming (non-reamed). This suggests that the choice of the initial fracture treatment strategy might be crucial for the overall outcome as well as for any post-traumatic cardiac consequences.

Fig 1. Alteration of connexin 43 distribution after multiple trauma in pigs.

A) Representative images of connexin 43 (Cx43) in the superficial and luminal layer of the left ventricle (LV) after multiple trauma and either nailing (non-reamed) or reaming. B) Amount of Connexin 43 measured in stained sections of the cardiac tissue after multiple trauma in pigs. Control/sham animals were presented as black bars, pigs after multiple trauma and nailing (non-reamed) were presented as light grey and pigs after multiple trauma and reaming (reamed) were presented as dark grey bars. C) Western blot analysis of Cx43 protein expression, as well as representative western blot bands. D) Systemic levels of connexin 43 in pg/mg measured in serum taken at baseline (BL), 4h and 6h after trauma. Baseline amounts are represented as white bars, 4 h after trauma as medium grey bars and 6 h after trauma as dark grey bars. E) Cx43 mRNA-expression in the LV presented as fold change. F) Connexin 40 (Cx40) mRNA expression detected in tissue homogenates of the left ventricle 6h after multiple trauma. G) Connexin 45 (Cx45) mRNA expression presented as fold change compared to sham. Results are significant (*) p<0.05. For statistical analysis one-way ANOVA was used. Graphical presentation as mean ± SEM. For each experiment n = 5.

Fig 2. Structural changes of the heart after multiple trauma.

A) Representative images of immunofluorescence staining of alpha actinin in the superficial and luminal layer of the left ventricle (LV) after multiple trauma and either nailing (non-reamed) or reaming (reamed) compared to sham. B) Quantitative analysis of alpha-actinin staining. Control/sham animals were presented as black bars, pigs after multiple trauma and nailing (Poly Nail, non-reamed) were presented as light grey and pigs after multiple trauma and reaming (reamed) were presented as dark grey bars. C) mRNA expression of alpha actinin presented as fold change compared to sham. D) Representative images of desmin immunofluorescence staining. E) Results of quantitative analysis of desmin staining in the superficial and luminal layer of left ventricles after multiple trauma. F) Desmin mRNA expression presented as fold change. G) Desmin mRNA expression in HL-1 cells in presence of PTC. H) Cx43 mRNA expression of HL-1 cells after incubation with PTC. I) mRNA expression of alpha-actinin in HL-1 cells after incubation with a defined polytrauma cocktail. Results are significant (*) p<0.05. For statistical analysis one-way ANOVA was used. Graphical presentation as mean ± SEM. For each experiment n = 5.

Fig 3. Cardiac inflammation.

Control/sham animals were presented in black bars, pigs after multiple trauma and nailing (non-reamed) were presented in light grey and pigs after multiple trauma and reaming (reamed) were presented as dark grey bars. A) mRNA expression of interleukin 1-ß (IL-1ß) of the luminal and superficial layer of the left ventricle, detected by RT-qPCR presented as fold change. B) Interleukin-6 (IL-6) protein expression and representative western blot bands. C) mRNA expression of IL-6 in the left ventricle. n = 5 for each experiment. Results are significant (*) p<0.05. For statistical analysis one-way ANOVA was used. Graphical presentation as mean ± SEM.

Fig 4. Nitrosative stress, cardiac tissue damage and apoptosis.

A) Representative images of the nitrotyrosine staining of the superficial and luminal layer of the left ventricle (LV) after multiple trauma and either nailing (non-reamed) or reaming (Poly Conv, reamed) compared to sham. B) Quantitative analysis of nitrotyrosine staining of the LV. Control/sham animals are presented as black bars, pigs after multiple trauma and nailing (Poly Nail, non-reamed) are presented as light grey and pigs after multiple trauma and reaming (reamed) are presented as dark grey bars. C) Systemic levels of HMGB1 in ng/ml measured by ELISA taken at baseline (BL), 4 h and 6 h after trauma. Baseline amounts are represented as white bars, 4 h after trauma as medium grey bars and 6 h after trauma as dark grey bars. D) Systemic levels of extracellular histones in μg/ml measured by ELISA at baseline (BL), 4 h and 6 h after trauma. Baseline amounts are represented as white bars, 4 h after trauma as medium grey bars and 6 h after trauma as dark grey bars. In each group n = 5. Results are significant (*) p<0.05. For statistical analysis one-way ANOVA was used. Graphical presentation as mean ± SEM.

Fig 5. Calcium balance in vivo and in vitro.

A) mRNA expression of Ryanodine receptor (RyR1) in tissue homogenates of the superficial and luminal layer of the left ventricle after multiple trauma in pigs. Control/sham animals were presented as black bars, pigs after multiple trauma and nailing (non-reamed), were presented as light grey and pigs after multiple trauma and reaming (reamed) were presented as dark grey bars. B) Sarcoplasmic/ endoplasmic reticulum ATPase (SERCA) mRNA expression after multiple trauma in pigs presented as fold change. C) mRNA expression of natrium calcium exchanger (NCX) presented as fold change. D) RyR1 mRNA expression of HL-1 cells in presence of polytrauma cocktail (PTC). E) SERCA mRNA expression of HL-1 cells incubated with PTC compared to control cells presented as fold change. F) mRNA expression of NCX in HL-1 cells in presence of PTC presented as fold change. Results are significant (*) p<0.05. For statistical analysis one-way ANOVA was used. Graphical presentation as mean ± SEM. For in vitro analysis students t-test was used (in vivo n = 5, in vitro n = 6).

Fig 6. In vitro analysis of HL-1 cells treated with polytrauma cocktail (PTC).

A) Troponin I level in supernatant of HL-1 cells in presence of PTC presented as ng/ml. B) mRNA expression of troponin I presented as fold change. C) Amount of heart fatty acid binding protein (HFABP) in supernatant of HL-1 cells after incubation with PTC presented as ng/ml. D) Cell viability in counts/sec in presence of PTC compared to control (PBS). E) mRNA expression of NLRP3 detected by RT-qPCR presented as fold change. mRNA expression of TLR-2 (F), -4 (G) and -9 (H) in presence of PTC presented as fold change. Results are significant (*) p<0.05. Graphical presentation as mean ± SEM. For statistical analysis students t-test was used. For all experiments n = 6.